HIV-1 continues to spread globally and no vaccine is available. Hence there is an urgent public health need for an effective microbicide to prevent sexual transmission of HIV-1. The topical application of potent combinations of viral fusion and entry inhibitors has been shown to exhibit microbicidal efficacy in the rhesus macaque vaginal transmission model. Peptides derived from the N- and C-terminal regions of the gp41 ectodomain (called N- and C-peptides, respectively) inhibit HIV-1 entry. N-peptides have generally proven far less potent than C-peptides. However, stabilization of a trimeric coiled-coil conformation of N peptides has been demonstrated to be a viable strategy to develop them as a new class of potent HIV-1 fusion inhibitors. We have recently identified and determined the crystal structure of an autonomously folded, N-peptide coiled-coil domain. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of this novel coiled-coil domain, and to use this knowledge to design and produce a bacterially expressed N-peptide fusion inhibitor for inclusion in a HIV-1 topical microbicide. Our central hypothesis is that the synergistic inhibition of different stages of the viral fusion and entry process can offer a powerful benefit in formulating an efficacious and more economic microbicide product. Specific aims of this research are: (1) to use modern protein engineering methods to identify and develop stabilized variants of a trimeric coiled-coil domain that display potent inhibitory activity against HIV-1 membrane fusion. We will use isoleucine- and valine-scanning mutagenesis to identify and incorporate specific residue substitutions that increase both trimer stability and antiviral potency. We will also construct chimeric N-peptides by using an isoleucine-zipper sequence, to stabilize the coiled-coil structure. Our emphasis is to generate potent N-peptide inhibitors suitable for development as an inexpensive component of a microbicide formulation. (2) To characterize the specificity, potency, and toxicity of improved N-peptide variants and their in vitro synergistic interactions with the virus-cell attachment inhibitors CMPD167 and BMS-378806, and the C-peptide fusion inhibitor C52L. We will conduct in vitro studies to determine inhibitory activity of select N-peptide variants against diverse primary HIV-1 isolates, and their toxic or inflammatory effects using the rabbit vaginal irritation model and in human cells. We will also study synergistic antiviral effects in vitro in order to make rational predictions for lead inhibitor combinations for formulation and efficacy testing in rhesus macaques. (3) To use the rhesus macaque high dose vaginal transmission model to assess the in vivo potency of an optimized N-peptide inhibitor alone and in combination with CMPD167, BMS- 378806, and C52L. We will evaluate the protection of macaques from vaginal challenge with both CCR5 and CXCR4 SHIVs by a vaginally delivered N-peptide inhibitor alone and in synergistic combination with CMPD167, BMS-378806, and C52L. [unreadable] [unreadable] [unreadable]